Acupuncture Effects on Neurotransmitter Metabolism

First, the rats were anesthetized with 2% isoflurane. Subsequently, a needle was inserted into ST36 or PC6, and a microdialysis probe (Eicom, Japan) was implanted between ST36 and ST37 or between PC6 and PC7. The samples were collected 30 min after the implantation of the probe (baseline, preacupuncture period). Then, acupuncture needles were inserted into bilateral ST36 and ST37 or PC6 and PC7 (ipsilateral as a pair), and 2 Hz EA was applied for 30 min. The samples were collected simultaneously during the EA period (acupuncture period). EA stimulation was stopped, and the needles were removed. Subsequently, the samples were collected for 30 min (postacupuncture period). The perfusion rate was set at 1.5 μL/min. The samples were placed in a box with ice cubes to prevent protease activation and then stored at −80°C until they were used for the metabolomic analysis of neurotransmitters (Figures 1(a) and 1(b)).
Finally, the rats were deeply anesthetized with 5% isoflurane, and the microdialysis probe was removed. A blood sample was collected from the heart, and a CSF sample was collected from the cisterna magna for the metabolomic analysis of neurotransmitters. Furthermore, samples of muscles surrounding the acupoints were collected for Western blot analysis (Figure 2).